PHOTOCHEMICAL REDUCTION AND OXIDATION OF MENADIONE (VITAMIN K3) IN CHLOROPLASTS

1. Menadione (vitamin K³) was found to be completely reduced byilluminated spinach chloroplasts under highly anaerobic conditionand in the presence of ethylenediamine tetraacetate (EDTA) inthe reaction mixture. This photoreductive reaction is sensitivetoward heat-treatment and inhibited by 2?10^-3M hydroxylamine.
2. In the presence of oxygen, the reduced form of menadione israpidly photooxidized by chloroplasts. This photooxidative activityalso is suppressed by heat-treatment but not inhibited by hydroxylamine.
3. Dyes which are inefficient as HILL oxidants such as thionineand methylene blue were found to be readily reduced by illuminatedchloroplasts, if the experimental conditions were appropriateto prevent the reoxidation of the photoreduced dyes; i.e., exhaustiveremoval of oxygen and the addition of EDTA in the reaction mixture.Menadione was found to accelerate the HILL reaction with thesedyes as oxidant under such experimental conditions.
4. In thepresence of molecular oxygen in the reaction mixture,menadionewas found to inhibit the HILL reaction with 2,6–dichlorophenolindophenol as oxidant, while the reaction rate was little influencedin high anaerobiosis.
5. These findings are explained by theintermediary oxidation and(photo-) reduction of menadione asan intermediary hydrogencarrier, and by the trends toward rapidphotooxidation of reducedmenadione.

(Received July 2, 1960; )     

METHYLENE BLUE INHIBITION OF PHOTOSYNTHESIS IN RHODOPSEUDOMONAS PALUSTRIS

1. Photosynthesis in Rhodopseudomonas palustris was found tobe completely inhibited by the reduced form of methylene blueat the concentration below 1x10^–5 M, provided that thecells had been in contact with the dye in the dark in anaerobiosis.The inhibition could be eliminated by washing the poisoned cellsor by replacing the anaerobic atmosphere with an aerobic one. 2. Other thiazine or oxazine dyes with E¹₀ near 0 volt, suchas thionine, brilliant cresyl blue and toluidine blue, in theirreduced forms, were also almost equally effective. 3. The mechanism of this dye-inhibition of bacterial photosynthesiswas discussed. ¹Present address: Laboratory of Biological Chemistry, TokyoInstitute of Technology, Meguro-ku, Tokyo. (Received August 21, 1961; )     

HYDROGENASE REACTIONS IN RHODOPSEUDOMONAS PALUSTRIS

1. The hydrogenase reactions in a purple non-sulfur bacterium,Rhodopseudornonas palustris, were investigated. Under photoheterotrophicculturing conditions, the photosynthetic activity of the cellswas found to be closely paralleled by the activity of hydrogenase.It was also revealed that the bacterium can grow under suchconditions even withont both photosynthetic and the enzyme activities. 2. The enzyme was revealed to be localized in the particulatefraction (presumably chromatophores) of the disintegrated cells.The properties of the enzyme in the cell-free preparation andin intact cells were described. 3. Among various hydrogen acceptors tested, p-benzoquinone wasmost rapidly hydrogenated. Some heat-labile factor was shownto be involved in the reduction of quinone, which was not requiredin the reduction of methylene blue. 4. The reactions of hydrogenase, both in cell-free state (quinone-and MB-reduction) and in intact cells (oxyhydrogen reaction),were markedly inhibited by molecular oxygen. The inhibitionwas noncompetitive with respect to H₂. A reversible mole-to-molecombination of the enzyme and O₂ was suggested as the mechanismof the inhibition. 5. Carbon monoxide inhibition was suggested also to be causedby a reversible mole-to-mole combination of the enzyme and theinhibitor. This inhibition was competitive with respect to H₂. 6. Rhodopseudomonas palustris hydrogenase was found to be refractorytoward cyanide, azide and sulfhydryl reagents. 7. Light markedly suppressed the oxyhydrogen reaction (intactcells) whereas other hydrogenation reactions (intact cells andcell- free preparations) were not affected by illumination. ¹Present address: Laboratory of Biological Chemistry, TokyoInstitute of Technology, Meguro-ku, Tokyo. (Received August 21, 1961; )     

STIMULATORY EFFECT OF CARBON DIOXIDE UPON THE HILL REACTION AS OBSERVED WTTH THE ADDITION OF CARBONIC ANHYDRASE TO REACTION MIXTURE

The stimulatory effect of CO₂ upon the HILL reaction by isolatedchloroplasts was observed with erythrocyte carbonic anhydraseas a supplementary agent for CO₂ deprivation. Addition of thisenzyme to the reaction media remarkably shortened the time requiredto obtain the maximal effect of CO₂ The degree of stimulationwas rather small (below 50 per cent) and varied depending onthe preparation of chloroplasts. In general, the effect wasgreater with broken chioroplasts than with whole chloroplasts.The lowering of light intensity diminished the CO₂-effect. ¹ Present address: Laboratory of Biological Chemistry, TokyoInstitute of Technology, Meguro-ku, Tokyo. (Received April 6, 1962; )